Multiple Labeling with MinX antibodies
Selection of antibodies for simultaneous detection of more than one antigen depends on at least two important criteria:
A) Availability of secondary antibodies that do not recognize
1.) one another i.e. are derived from the same host species,
2.) other primary antibodies used in the assay system,
3.) immunoglobulins from other species present in the assay system, or
4.) endogenous immunoglobulins present in the tissues or cells under investigation.
B) Use of labels (fluorophores, enzyme-reaction products, electron-dense particles) that are well resolved.
The affinity-purified antibodies marked MinX (minimal cross-reactivity/reaction) have been specifically prepared to meet these criteria. They have been tested and/or adsorbed against IgG and/or serum proteins from other species.
Their cross-reactivity (X) with immunoglobulins of those indicated species is “minimal” (Min), which means below < 1%.
(Ha = Armenian Hamster, Hs = Syrian Hamster, Ck = Chicken, Hu = Human, Rb = Rabbit, Ms = Mouse, Gp = Guinea Pig, Ho = Horse, Rt = Rat, Bo = Bovine, Sh = Sheep, Sw = Swine, Go = Goat)
One of many possible multiple-labeling protocols using these reagents with minimal cross-reaction is shown in the following example. In this example, the secondary antibodies used in step 3 do not recognize each other since they are all made in donkey. They have been solid-phase adsorbed so that they do not recognize the other primary antibodies used in Step 2. Also, they do not react with endogenous rat immunoglobulins, which may be present in the rat tissue. However, the increase in specificity by adsorption to Ig/ serum proteins is at the expenses of the sensitivity: secondary antibodies from donkey, which are adsorbed against up to ten different species are less affine than secondary antibodies from goat, which have been adsorbed against Ig/ serum proteins from only three to five different species.
Note: Antibodies, which have been adsorbed against a closely related species, partly show a reduced epitope-recognition, so that monoclonal primary antibodies with rare IgG isotypes (IgG2b, IgG3), which are largely homologous to the species used for adsorption, may not be recognized as well any more.
Triple Labeling of Rat Tissue
* Also see: Example Fab blocking of tissue
Note: The detection of antigen A, B and C can be carried out simultaneously or consecutively. In a parallel approach, the antibodies in step 2 (b, d, f) and 3 (c, e, g) are applied as a cocktail, in the consecutive approach all single steps from a) to g) are done in successive incubations. In both approaches thorough washing between the antibody incubations and after blocking (a) is required. With heavy or persistent background in the consecutive approach, further blocking may be required before Steps d) and f). The multiple staining can be carried out in a parallel approach, if the staining and background characteristics of the antibodies after mixture are unchanged compared to single (or consecutive) stains with these antibodies. In mixtures, antibodies (immunoglobulins) may react with each other because of unspecific interactions and thereby may interfere with the antigen detection. For this reason no normal serum should be added to the antibody dilution buffer.
More Tips and Tricks:
For a review of multi-color immunofluorescence labeling with confocal microscopy see Brelje, Wessendorf, and Sorenson, “Multi-color laser scanning confocal immunofluorescence microscopy: Practical application and limitations.” In Cell Biological Applications of Confocal Microscopy, B. Matsumoto. Orlando, FL: Academic Press, Inc. (Methods Cell. Biol. 1993, Vol. 38, 97; Methods Cell. Biol. 2002, Vol. 70, 165).